Sequencing trait-associated mutations to clone wheat rust-resistance gene YrNAM.

Stripe (yellow) rust, caused by Puccinia striiformis f. sp. tritici (Pst), can significantly affect wheat production. Cloning resistance genes is critical for efficient and effective breeding of stripe rust resistant wheat cultivars. One resistance gene (Yr10CG) underlying the Pst resistance locus Yr10 has been cloned. However, following haplotype and linkage analyses indicate the presence of additional Pst resistance gene(s) underlying/near Yr10 locus. Here, we report the cloning of the Pst resistance gene YrNAM in this region using the method of sequencing trait-associated mutations (STAM). YrNAM encodes a non-canonical resistance protein with a NAM domain and a ZnF-BED domain. We show that both domains are required for resistance. Transgenic wheat harboring YrNAM gene driven by its endogenous promoter confers resistance to stripe rust races CYR32 and CYR33. YrNAM is an ancient gene and present in wild wheat species Aegilops longissima and Ae. sharonensis; however, it is absent in most wheat cultivars, which indicates its breeding value.

The Emergence of Fusarium oxysporum f. sp. apii Race 4 and Fusarium oxysporum f. sp. coriandrii Highlights Major Obstacles Facing Agricultural Production in Coastal California in a Warming Climate: A Case Study.

Currently, Fusarium oxysporum f. sp. apii (Foa) race 4 in celery and F. oxysporum f. sp. coriandrii (Foci) in coriander have the characteristics of emerging infectious plant diseases in coastal southern California: the pathogens are spreading, yield losses can be severe, and there are currently no economical solutions for their control. Celery, and possibly coriander, production in these regions is are likely to have more severe disease from projected warmer conditions in the historically cool, coastal regions. Experimental evidence shows that Foa race 4 causes much higher disease severity when temperatures exceed 21°C. A phylogenomic analysis indicated that Foa race 4, an older, less virulent, and uncommon Foa race 3, and two Foci are closely related in their conserved genomes. These closely related genotypes are somatically compatible. Foa race 4 can also cause disease in coriander and the two organisms readily form "hetero" conidial anastomosis tubes (CAT), further increasing the likelihood of parasexual recombination and the generation of novel pathotypes. A horizontal chromosome transfer event likely accounts for the difference in host range between Foci versus Foa races 4 and 3 because they differ primarily in one or two accessory chromosomes. How Foa race 4 evolved its hyper-virulence is unknown. Although the accessory chromosomes of Foa races 3 and 4 are highly similar, there is no evidence that Foa race 4 evolved directly from race 3, and races 3 and 4 probably only have a common ancestor. Foa race 2, which is in a different clade within the Fusarium oxysporum species complex (FOSC) than the other Foa, did not contribute to the evolution of race 4, and does not form CATs with Foa race 4; consequently, while inter-isolate CAT formation is genetically less restrictive than somatic compatibility, it might be more restricted between FOSC clades than currently known. Other relatively new F. oxysporum in coastal California include F. oxysporum f. sp. fragariae on strawberry (Fof). Curiously, Fof "yellows-fragariae" isolates also have similar core genomes to Foa races 4 and 3 and Foci, perhaps suggesting that there may be core genome factors in this lineage that favor establishment in these soils.

The Effect of Temperature on Disease Severity and Growth of Fusarium oxysporum f. sp. apii Races 2 and 4 in Celery.

Fusarium oxysporum f. sp. apii race 4, which is in F. oxysporum species complex (FOSC) Clade 2, causes a new Fusarium wilt of celery. We compared F. oxysporum f. sp. apii race 4 with race 2, which causes Fusarium yellows of celery and is in FOSC Clade 3. Optimal temperatures for celery yield are 16 to 18°C. Soil temperatures in California celery production areas can range up to 26°C, and the maximal rate of hyphal extension of F. oxysporum f. sp. apii races 2 and 4 in culture are 25 and 28°C, respectively. Here, we compared the effect of temperatures from 16 to 26°C on growth of F. oxysporum f. sp. apii races 4 and 2 in two celery cultivars: Challenger, which is resistant to F. oxysporum f. sp. apii race 2 and susceptible to race 4; and Sonora, which is susceptible to both F. oxysporum f. sp. apii races 2 and 4. Based on linear regressions, as temperature increases, there is an increase in the log of F. oxysporum f. sp. apii race 4 DNA concentration in celery crowns and in the reduction in plant height. Based on logistic regressions, as temperature increases, the incidence of vascular discoloration increases in celery with either F. oxysporum f. sp. apii race 2 or 4 infection. In both cultivars, temperatures of 22°C and above resulted in a significantly (α = 0.05) greater concentration of F. oxysporum f. sp. apii race 4 than race 2 in planta. The concentration of F. oxysporum f. sp. apii race 2 in crowns in 'Challenger' is temperature-independent and comparatively low; consequently, 'Challenger' is, at least partly, resistant rather than tolerant to F. oxysporum f. sp. apii race 2.

Horizontal chromosome transfer and independent evolution drive diversification in Fusarium oxysporum f. sp. fragariae.

The genes required for host-specific pathogenicity in Fusarium oxysporum can be acquired through horizontal chromosome transfer (HCT). However, it is unknown if HCT commonly contributes to the diversification of pathotypes. Using comparative genomics and pathogenicity phenotyping, we explored the role of HCT in the evolution of F. oxysporum f. sp. fragariae, the cause of Fusarium wilt of strawberry, with isolates from four continents. We observed two distinct syndromes: one included chlorosis ('yellows-fragariae') and the other did not ('wilt-fragariae'). All yellows-fragariae isolates carried a predicted pathogenicity chromosome, 'chrY-frag ', that was horizontally transferred at least four times. chrY-frag was associated with virulence on specific cultivars and encoded predicted effectors that were highly upregulated during infection. chrY-frag was not present in wilt-fragariae; isolates causing this syndrome evolved pathogenicity independently. All origins of F. oxysporum f. sp. fragariae occurred outside of the host's native range. Our data support the conclusion that HCT is widespread in F. oxysporum, but pathogenicity can also evolve independently. The absence of chrY-frag in wilt-fragariae suggests that multiple, distinct pathogenicity chromosomes can confer the same host specificity. The wild progenitors of cultivated strawberry (Fragaria × ananassa) did not co-evolve with this pathogen, yet we discovered several sources of genetic resistance.

Phylogenomic Analysis of a 55.1-kb 19-Gene Dataset Resolves a Monophyletic Fusarium that Includes the Fusarium solani Species Complex.

Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. In 2013, the Fusarium community voiced near unanimous support for a concept of Fusarium that represented a clade comprising all agriculturally and clinically important Fusarium species, including the F. solani species complex (FSSC). Subsequently, this concept was challenged in 2015 by one research group who proposed dividing the genus Fusarium into seven genera, including the FSSC described as members of the genus Neocosmospora, with subsequent justification in 2018 based on claims that the 2013 concept of Fusarium is polyphyletic. Here, we test this claim and provide a phylogeny based on exonic nucleotide sequences of 19 orthologous protein-coding genes that strongly support the monophyly of Fusarium including the FSSC. We reassert the practical and scientific argument in support of a genus Fusarium that includes the FSSC and several other basal lineages, consistent with the longstanding use of this name among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, students, and researchers with a stake in its taxonomy. In recognition of this monophyly, 40 species described as genus Neocosmospora were recombined in genus Fusarium, and nine others were renamed Fusarium. Here the global Fusarium community voices strong support for the inclusion of the FSSC in Fusarium, as it remains the best scientific, nomenclatural, and practical taxonomic option available.

A haplotype-phased genome of wheat stripe rust pathogen Puccinia striiformis f. sp. tritici, race PST-130 from the Western USA.

More virulent and aggressive races of Puccinia striiformis f. sp. tritici (Pst), the pathogen causing wheat stripe rust, have been spreading around the world since 2000 causing large grain yield losses. A better understanding of the genome and genetic diversity of these new Pst races will be useful to develop new strategies to ameliorate these losses. In this study, we generated an improved genome assembly of a post-2000 virulent race from the Western USA designated as PST-130. We implemented a haplotype phasing strategy using the diploid-aware assembler, Falcon-Unzip and new long-read technology from PacBio to phase the two genomes of this dikaryotic organism. The combination of these new technologies resulted in an improved PST-130 assembly with only 151 contigs (85.4 Mb, N50 of 1.44 Mb), and a complementary assembly (haplotigs) with 458 contigs (65.9 Mb, N50 of 0.235 Mb, PRJNA650506). This new assembly improved gene predictions resulting in 228 more predicted complete genes than in the initial Illumina assembly (29,178 contigs, N50 of 5 kb). The alignment of the non-repetitive primary and haplotig contigs revealed and average of 5.22 SNP/kb, with 39.1% showing <2 SNP/kb and 15.9% >10 SNP/kb. This large divergent regions may represent introgressions of chromosome segments from more divergent Pst races in regions where a complete sexual cycle and recombination are possible. We hypothesize that some of the divergent regions in PST-130 may be related to the European "Warrior" race PST-DK0911 because this genome is more similar to PST-130 (3.18 SNP/kb) than to the older European race PST-104E (3.75 SNP/kb). Complete phasing of additional Pst genomes or sequencing individual nuclei will facilitate the tracing of the haploid genomes introduced by the new Pst races into local populations.

Genomic differences between the new Fusarium oxysporum f. sp. apii (Foa) race 4 on celery, the less virulent Foa races 2 and 3, and the avirulent on celery f. sp. coriandrii.

BACKGROUND: Members of the F. oxysporium species complex (FOSC) in the f. sp. apii (Foa) are pathogenic on celery and those in f. sp. coriandrii (Foci) are pathogenic on coriander (=cilantro). Foci was first reported in California in 2005; a new and highly aggressive race 4 of Foa was observed in 2013 in California. Preliminary evidence indicated that Foa can also cause disease on coriander, albeit are less virulent than Foci. Comparative genomics was used to investigate the evolutionary relationships between Foa race 4, Foa race 3, and the Foci, which are all in FOSC Clade 2, and Foa race 2, which is in FOSC Clade 3. RESULTS: A phylogenetic analysis of 2718 single-copy conserved genes and mitochondrial DNA sequence indicated that Foa races 3 and 4 and the Foci are monophyletic within FOSC Clade 2; these strains also are in a single somatic compatibility group. However, in the accessory genomes, the Foci versus Foa races 3 and 4 differ in multiple contigs. Based on significantly increased expression of Foa race 4 genes in planta vs. in vitro, we identified 23 putative effectors and 13 possible pathogenicity factors. PCR primers for diagnosis of either Foa race 2 or 4 and the Foci were identified. Finally, mixtures of conidia that were pre-stained with different fluorochromes indicated that Foa race 4 formed conidial anastomosis tubes (CATs) with Foci. Foa race 4 and Foa race 2, which are in different somatic compatibility groups, did not form CATs with each other. CONCLUSIONS: There was no evidence that Foa race 2 was involved in the recent evolution of Foa race 4; Foa race 2 and 4 are CAT-incompatible. Although Foa races 3 and 4 and the Foci are closely related, there is no evidence that either Foci contributed to the evolution of Foa race 4, or that Foa race 4 was the recent recipient of a multi-gene chromosomal segment from another strain. However, horizontal chromosome transfer could account for the major difference in the accessory genomes of Foa race 4 and the Foci and for their differences in host range.

An ancestral NB-LRR with duplicated 3'UTRs confers stripe rust resistance in wheat and barley.

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a global threat to wheat production. Aegilops tauschii, one of the wheat progenitors, carries the YrAS2388 locus for resistance to Pst on chromosome 4DS. We reveal that YrAS2388 encodes a typical nucleotide oligomerization domain-like receptor (NLR). The Pst-resistant allele YrAS2388R has duplicated 3' untranslated regions and is characterized by alternative splicing in the nucleotide-binding domain. Mutation of the YrAS2388R allele disrupts its resistance to Pst in synthetic hexaploid wheat; transgenic plants with YrAS2388R show resistance to eleven Pst races in common wheat and one race of P. striiformis f. sp. hordei in barley. The YrAS2388R allele occurs only in Ae. tauschii and the Ae. tauschii-derived synthetic wheat; it is absent in 100% (n = 461) of common wheat lines tested. The cloning of YrAS2388R will facilitate breeding for stripe rust resistance in wheat and other Triticeae species.

Cloning of the Cytochrome b Gene From the Tomato Powdery Mildew Fungus Leveillula taurica Reveals High Levels of Allelic Variation and Heteroplasmy for the G143A Mutation.

Leveillula taurica is a major pathogen of tomato and several other crops that can cause substantial yield losses in favorable conditions for the fungus. Quinone outside inhibitor fungicides (QoIs) are routinely used for the control of the pathogen in tomato fields across California, but their recurrent use could lead to the emergence of resistance against these compounds. Here, we partially cloned the cytochrome b gene from L. taurica (Lt cytb) and searched within populations of the fungus collected from tomato fields across California for mutations that confer resistance to QoIs. A total of 21 single nucleotide polymorphisms (SNPs) were identified within a 704 bp fragment of the Lt cytb gene analyzed, of which five were non-synonymous substitutions. Among the most frequent SNPs encountered within field populations of the pathogen was the G143A substitution that confers high levels of resistance against QoIs in several fungi. The other four amino acid substitutions were novel mutations, whose effect on QoI resistance is currently unknown. Sequencing of the Lt cytb gene from individual single-cell conidia of the fungus further revealed that most SNPs, including the one leading to the G143A substitution, were present in a heteroplasmic state, indicating the co-existence of multiple mitotypes in single cells. Analysis of the field samples showed that the G143A substitution is predominantly heteroplasmic also within field populations of L. taurica in California, suggesting that QoI resistance in this fungus is likely to be quantitative rather than qualitative.

Wheat Ms2 encodes for an orphan protein that confers male sterility in grass species.

Male sterility is a valuable trait for plant breeding and hybrid seed production. The dominant male-sterile gene Ms2 in common wheat has facilitated the release of hundreds of breeding lines and cultivars in China. Here, we describe the map-based cloning of the Ms2 gene and show that Ms2 confers male sterility in wheat, barley and Brachypodium. MS2 appears as an orphan gene within the Triticinae and expression of Ms2 in anthers is associated with insertion of a retroelement into the promoter. The cloning of Ms2 has substantial potential to assemble practical pipelines for recurrent selection and hybrid seed production in wheat.

Races of the Celery Pathogen Fusarium oxysporum f. sp. apii Are Polyphyletic.

Fusarium oxysporum species complex (FOSC) isolates were obtained from celery with symptoms of Fusarium yellows between 1993 and 2013 primarily in California. Virulence tests and a two-gene dataset from 174 isolates indicated that virulent isolates collected before 2013 were a highly clonal population of F. oxysporum f. sp. apii race 2. In 2013, new highly virulent clonal isolates, designated race 4, were discovered in production fields in Camarillo, California. Long-read Illumina data were used to analyze 16 isolates: six race 2, one of each from races 1, 3, and 4, and seven genetically diverse FOSC that were isolated from symptomatic celery but are nonpathogenic on this host. Analyses of a 10-gene dataset comprising 38 kb indicated that F. oxysporum f. sp. apii is polyphyletic; race 2 is nested within clade 3, whereas the evolutionary origins of races 1, 3, and 4 are within clade 2. Based on 6,898 single nucleotide polymorphisms from the core FOSC genome, race 3 and the new highly virulent race 4 are highly similar with Nei's Da = 0.0019, suggesting that F. oxysporum f. sp. apii race 4 evolved from race 3. Next generation sequences were used to develop PCR primers that allow rapid diagnosis of races 2 and 4 in planta.

Wheat Stripe Rust Resistance Protein WKS1 Reduces the Ability of the Thylakoid-Associated Ascorbate Peroxidase to Detoxify Reactive Oxygen Species.

Stripe rust is a devastating fungal disease of wheat caused by Puccinia striiformis f. sp tritici (Pst). The WHEAT KINASE START1 (WKS1) resistance gene has an unusual combination of serine/threonine kinase and START lipid binding domains and confers partial resistance to Pst. Here, we show that wheat (Triticum aestivum) plants transformed with the complete WKS1 (variant WKS1.1) are resistant to Pst, whereas those transformed with an alternative splice variant with a truncated START domain (WKS1.2) are susceptible. WKS1.1 and WKS1.2 preferentially bind to the same lipids (phosphatidic acid and phosphatidylinositol phosphates) but differ in their protein-protein interactions. WKS1.1 is targeted to the chloroplast where it phosphorylates the thylakoid-associated ascorbate peroxidase (tAPX) and reduces its ability to detoxify peroxides. Increased expression of WKS1.1 in transgenic wheat accelerates leaf senescence in the absence of Pst. Based on these results, we propose that the phosphorylation of tAPX by WKS1.1 reduces the ability of the cells to detoxify reactive oxygen species and contributes to cell death. This response takes several days longer than typical hypersensitive cell death responses, thus allowing the limited pathogen growth and restricted sporulation that is characteristic of the WKS1 partial resistance response to Pst.

Host-induced gene silencing inhibits the biotrophic pathogen causing downy mildew of lettuce.

Host-induced gene silencing (HIGS) is an RNA interference-based approach in which small interfering RNAs (siRNAs) are produced in the host plant and subsequently move into the pathogen to silence pathogen genes. As a proof-of-concept, we generated stable transgenic lettuce plants expressing siRNAs targeting potentially vital genes of Bremia lactucae, a biotrophic oomycete that causes downy mildew, the most important disease of lettuce worldwide. Transgenic plants, expressing inverted repeats of fragments of either the Highly Abundant Message #34 (HAM34) or Cellulose Synthase (CES1) genes of B. lactucae, specifically suppressed expression of these genes, resulting in greatly reduced growth and inhibition of sporulation of B. lactucae. This demonstrates that HIGS can provide effective control of B. lactucae in lettuce; such control does not rely on ephemeral resistance conferred by major resistance genes and therefore offers new opportunities for durable control of diverse diseases in numerous crops.

Endogenous ergothioneine is required for wild type levels of conidiogenesis and conidial survival but does not protect against 254 nm UV-induced mutagenesis or kill.

Ergothioneine, a histidine derivative, is concentrated in conidia of ascomycetous fungi. To investigate the function of ergothioneine, we crossed the wild type Neurospora crassa (Egt(+)) and an ergothioneine non-producer (Egt(-), Δegt-1, a knockout in NCU04343.5) and used the Egt(+) and Egt(-) progeny strains for phenotypic analyses. Compared to the Egt(+) strains, Egt(-) strains had a 59% reduction in the number of conidia produced on Vogel's agar. After storage of Egt(+) and Egt(-) conidia at 97% and 52% relative humidity (RH) for a time course to either 17 or 98 days, respectively, Egt(-) strains had a 23% and a 18% reduction in life expectancy at 97% and 52% RH, respectively, compared to the Egt(+) strains. Based on a Cu(II) reduction assay with the chelator bathocuproinedisulfonic acid disodium salt, ergothioneine accounts for 38% and 33% of water-soluble antioxidant capacity in N. crassa conidia from seven and 20 day-old cultures, respectively. In contrast, ergothioneine did not account for significant (α=0.05) anti-oxidant capacity in mycelia, which have lower concentrations of ergothioneine than conidia. The data are consistent with the hypothesis that ergothioneine has an antioxidant function in vivo. In contrast, experiments on the spontaneous mutation rate in Egt(+) and Egt(-) strains and on the effects of 254 nm UV light on mutation rate and conidial viability do not support the hypothesis that ergothioneine protects DNA in vivo.

Fifty years since Silent Spring.

Rachel Carson's 1962 Silent Spring exposed both observed and potential environmental and health externalities of the increasing organochlorine and organophosphate insecticide use in the United States post-World War II. Silent Spring was a critical component in a popular movement that resulted in increased regulation and the development of safer pesticides. Most changes in pesticide use in the global north have involved pesticide substitutions, although riskier pesticides remain in use. Many ideas in Silent Spring are compatible with the theory of integrated pest management (IPM), and IPM has been broadly embraced in the United States and internationally as a strategy for achieving least-use and/or least-risk pesticide use in agriculture. IPM is a politically feasible policy that purports to reduce pesticide use and/or risk in agriculture but often does not, except in extreme cases of pesticide overuse that result in negative agricultural/economic consequences for growers.

Characterization of cognitive dysfunction in Sjögren's syndrome patients.

Sjögren's syndrome is an autoimmune disorder primarily affecting women, with decreased saliva and tear production as the principal characteristic. Cognitive, neurological, and psychiatric disorders also are associated with Sjögren's. The present study addressed the hypothesis that patients with Sjögren's syndrome differ significantly from matched controls in the prevalence and impact of a number of neuropsychiatric abnormalities. Sjögren's patients and controls (n = 37 per group) underwent medical and psychiatric evaluation, demographic assessments, quality of life and symptom evaluation, and extensive testing of cognitive function and memory. Patients and controls were closely matched for age, gender distribution, verbal IQ, marital status, educational level, employment status, and current/past medical or psychiatric history. On most subjective self-ratings, Sjögren's patients reported greater fatigue, impaired physical functioning, feeling depressed, and autonomic symptomatology compared to controls. Impaired memory was described mainly as loss of thought continuity in the midst of a task or activity. However, the majority of objective measures of cognition, psychomotor function, and memory showed minimal differences between groups. Sjögren's patients rate themselves as impaired on multiple ratings of emotional, cognitive, and physical function, but objective measures of cognition reveal fewer substantive differences between patients and matched controls. Sjögren's patients perceive deteriorated physical function over time, but they achieve a level of functioning comparable to controls despite the burden of their illness.

Clades of γ-glutamyltransferases (GGTs) in the ascomycota and heterologous expression of Colletotrichum graminicola CgGGT1, a member of the pezizomycotina-only GGT clade.

Gamma-glutamyltransferase (GGT, EC 2.3.2.2) cleaves the γ-glutamyl linkage in glutathione (GSH). Ascomycetes in either the Saccharomycotina or the Taphrinomycotina have one to three GGTs, whereas members of the Pezizomycotina have two to four GGTs. A Bayesian analysis indicates there are three well-supported main clades of GGTs in the Ascomycota. 1) A Saccharomycotina and a Taphrinomycotina-specific GGT sub-clade form a yeast main clade. This clade has the three relatively well-characterized fungal GGTs: (Saccharomyces cerevisiae CIS2 and Schizosaccharomyces pombe Ggt1 and Ggt2) and most of its members have all 14 of the highly conserved and critical amino acids that are found in GGTs in the other kingdoms. 2) In contrast, a main clade (GGT3) differs in 11 of the 14 highly conserved amino acids that are found in GGTs in the other kingdoms. All of the 44 Pezizomycotina analyzed have either one or two GGT3s. 3) There is a Pezizomycotina-only GGT clade that has two well-supported sub-clades (GGT1 and GGT2); this clade differs in only two of the 14 highly conserved amino acids found in GGTs in the other kingdoms. Because the Pezizomycotina GGTs differ in apparently critical amino acids from the cross-kingdom consensus, a putative GGT from Colletotrichum graminicola, a member of the Pezizomycotina, was cloned and the protein product was expressed as a secreted protein in Pichia pastoris. A GGT enzyme assay of the P. pastoris supernatant showed that the recombinant protein was active, thereby demonstrating that CgGGT1 is a bona fide GGT.

One fungus, one name: defining the genus Fusarium in a scientifically robust way that preserves longstanding use.

In this letter, we advocate recognizing the genus Fusarium as the sole name for a group that includes virtually all Fusarium species of importance in plant pathology, mycotoxicology, medicine, and basic research. This phylogenetically guided circumscription will free scientists from any obligation to use other genus names, including teleomorphs, for species nested within this clade, and preserve the application of the name Fusarium in the way it has been used for almost a century. Due to recent changes in the International Code of Nomenclature for algae, fungi, and plants, this is an urgent matter that requires community attention. The alternative is to break the longstanding concept of Fusarium into nine or more genera, and remove important taxa such as those in the F. solani species complex from the genus, a move we believe is unnecessary. Here we present taxonomic and nomenclatural proposals that will preserve established research connections and facilitate communication within and between research communities, and at the same time support strong scientific principles and good taxonomic practice.

γ-Glutamyltransferases (GGT) in Colletotrichum graminicola: mRNA and enzyme activity, and evidence that CgGGT1 allows glutathione utilization during nitrogen deficiency.

Gamma-glutamyltransferase (GGT, EC 2.3.2.2) cleaves the γ-glutamyl linkage in glutathione (GSH). Three GGTs in the hemibiotrophic plant pathogen Colletotrichum graminicola were identified in silico. GGT mRNA expression was monitored by quantitative reverse-transcriptase PCR. Expression of all three genes was detected in planta during the biotrophic and necrotrophic stages of infection. Of the three GGTs, CgGGT1 mRNA (from gene GLRG_09590) was the most highly expressed. All three GGT mRNAs were up-regulated in wild type nitrogen-starved germlings in comparison to non-starved germlings. CgGGT1 was insertionally mutagenized in C. graminicola, complemented with the wild type form of the gene, and over-expressed. Enzyme assays of two independent CgGGT1 knockouts and the wild type indicated that CgGGT1 is the major GGT and accounts for 86% and 68% of total GGT activity in conidia and mycelia, respectively. The over-expressing strain had 8-fold and 3-fold more enzyme activity in conidia and mycelia, respectively, than the wild type. In an analysis of the GGT knockout, complemented and over-expressing strains, GGT1 transcript levels are highly correlated (r=0.95) with levels of total GGT enzyme activity. CgGGT1 and CgGGT2 genes in strains that had ectopic copies of CgGGT1 were not up-regulated by nitrogen-starvation, in contrast to the wild type. Deletion or over-expression of CgGGT1 had no effect on mRNA expression of CgGGT2 and CgGGT3. In broth in which 3 and 6mM glutathione (GSH) was the nitrogen source, the CgGGT1 over-expressing strain produced significantly (P<0.0001) more biomass than the wild type and complemented strains, whereas the CgGGT1Δ strains produced significantly (P<0.0001) less biomass than the wild type strain. This suggests that CgGGT1 is involved in utilizing GSH as a nitrogen source. However, deletion and over-expression of CgGGT1 had no effect on either virulence in wounded corn leaf sheaths or GSH levels in conidia and mycelia. Thus, the regulation of GSH concentration is apparently independent of CgGGT1 activity.

Lifestyle transitions in plant pathogenic Colletotrichum fungi deciphered by genome and transcriptome analyses.

Colletotrichum species are fungal pathogens that devastate crop plants worldwide. Host infection involves the differentiation of specialized cell types that are associated with penetration, growth inside living host cells (biotrophy) and tissue destruction (necrotrophy). We report here genome and transcriptome analyses of Colletotrichum higginsianum infecting Arabidopsis thaliana and Colletotrichum graminicola infecting maize. Comparative genomics showed that both fungi have large sets of pathogenicity-related genes, but families of genes encoding secreted effectors, pectin-degrading enzymes, secondary metabolism enzymes, transporters and peptidases are expanded in C. higginsianum. Genome-wide expression profiling revealed that these genes are transcribed in successive waves that are linked to pathogenic transitions: effectors and secondary metabolism enzymes are induced before penetration and during biotrophy, whereas most hydrolases and transporters are upregulated later, at the switch to necrotrophy. Our findings show that preinvasion perception of plant-derived signals substantially reprograms fungal gene expression and indicate previously unknown functions for particular fungal cell types.